The mechanism of immune tolerance to ACI heart allografts in high responder Lewis rats given a post-transplant protocol of total lymphoid irradiation (TLI), anti-thymocyte globin (ATG), and a donor blood transfusion will be studied. Preliminary results show that the mixed leukocyte reaction (MLR) of tolerant recipients to donor stimulator cells is normal as judged by proliferation, but the cytokine secretion pattern is abnormal and indicates a shift from a Th1 to a Th2 pattern. In addition, spleen cells from tolerant recipients were unable to rapidly reject donor hearts in irradiated adoptive hosts when injected after heart transplantation, but rapidly rejected donor grafts when injected 24 hours before transplantation. These results can be explained by the hypothesis that tolerance is due to a shift from Th1 to Th2 CD4+ T cells. Th1 cells remain in the tolerant recipient as long-lived memory cells and can reject grafts in adoptive hosts in the absence of Th2 cells. Th2 cells present in the tolerant recipients prevent rejection, are constantly activated by donor antigen, and are short-lived (die or inactivated within 24 hours after removal of antigen). In order to test this hypothesis, the pattern of Th1 and Th2 cytokine (IL- 2,IL-4,gamma-IFN,IL-10) secretion will be studied in the MLR from tolerant(TLI, ATG, blood cell transfusion) and non-tolerant (TLI and ATG only) recipients at serial time points after transplantation. Transient activation markers, identified by OX-40 antibodies will be studied simultaneously. In addition, graft biopsies from recipients will be monitored at serial time points for the transcript ion of Th1 and Th2 cytokine genes by in situ hybridization, and for the presence of transient activation markers. Finally, the subsets of donor cells which induce tolerance, and subsets of recipient cells which maintain tolerance will be identified in adoptive transfer studies.